Chronic myeloid leukemia (CML) is the first clinically cured stem cell-derived hematopoietic neoplasm; however, tyrosine kinase inhibitor (TKI) therapy leaves behind a pool of cells showing innate resistance to these drugs. These are quiescent CML stem cells (HSC) that represent an active cancer reservoir. Thus, it becomes clear that only drugs that can safely and efficiently target these HSCs without harming the normal ones have the potential to lead to disease eradication. Our published and preliminary data suggest that a likely mechanism involve an aberrant balance between kinases (i.e. BCR-ABL1 and Jak2) and phosphatases (i.e. PP2A) and this may depend on altered expression of specific microRNAs (miRs). In fact, we found altered expression of miRs that may target the Jak2-hnRNP A1-SET/PP2A-2-catenin HSC pathway, which is essential for survival and self-renewal of quiescent CML HSCs but operates in a BCR-ABL1 expression- but not kinase- dependent manner. Given that a) BCR-ABL1 protein but not mRNA and Jak2 expression/activity are elevated in quiescent CML HSCs; b) miR levels are aberrant in leukemias and change in stem vs. progenitor cells; and c) the recently discovered RNA decoy activity is not limited to miR-328 but other miRs likely interact and interfere with hnRNP function; the hypothesis driving this proposal is that quiescent Ph+ HSCs display a dysregulated miR expression which depends on BCR-ABL1 expression but not kinase activity, and contributes to enhanced survival and self-renewal of leukemic HSCs. Based on these considerations, the overall objective of this proposal is to understand the requirement of altered miR expression for the maintenance of the quiescent reservoir of CML HSCs and determine the therapeutic relevance of pharmacologic restoration of miR expression through the identification and integrated in vitro and in vivo analysis of the miRs, which in a canonical and/or decoy manner, affect CML HSC survival and self-renewal through direct interference with the BCR-ABL1-Jak2-hnRNP A1-SET-2-catenin pathway. Thus, to accomplish these goals we will rely on the use of highly HSC-enriched CD34+/CD38-(CD90+) BM cells form CML patients, the unique SCL-tTA-BCR/ABL mouse model and synthetic miRs or antagomiR to treat NSG mice transplanted with GFP+/Luc+ CML HSCs in order to: 1) Identify miRs dysregulated in CML HSCs that interfere with the BCR-ABL1-Jak2 pathway through their canonical and/or hnRNP A1 decoy activities; 2) Assess whether modulation of miR levels impairs in vitro and in vivo survival and self-renewal of CML HSCs; and 3) Determine the therapeutic role of modulation of miR expression in eradication of CML by using 2-O-MePS miRs and antagomiRs. We are confident that the successful completion of the proposed work will advance our knowledge of leukemic HSC biology and, based on the discoveries we made in the past few years, it is safe to predict that new observations will be made in this field and that some of them will reveal new strategies for therapeutic intervention. Hence, the strong importance and high relevance of this work for basic and translational cancer research.